Gene editing-based method of attenuating the beta-amyloid pathway

ABSTRACT

Described herein are CRISPR/Cas9 constructs designed for the C-terminal truncation of human amyloid precursor protein (APP) as well as methods of making and using such a construct. A Cas9 nuclease/gRNA ribonucleoprotein directs cleavage of an APP gene to provide a C-terminal truncated APP having a length of 659, 670, 676, or 686 amino acids, relative to the human or mouse APP sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/618,694, filed Jan. 18, 2018, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AG048218 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 18, 2019, isnamed 960296_02327_ST25.txt and is 101,371 bytes in size.

BACKGROUND

The gradual accumulation of Aβ in brains is a neuropathologic hallmarkof Alzheimer's disease (AD). Aβ is generated by the sequential cleavageof the amyloid precursor protein (APP) by β- and γ-secretases(β-secretase aka BACE-1, and γ-secretase), with BACE-1-cleavage as therate-limiting step. Substantial evidence indicates that accrual ofAPP-cleavage products play a key role in AD, making the “amyloidogenicpathway” an important therapeutic target (1-3).

CRISPR/Cas9 gene editing is emerging as a promising tool to disrupt theexpression of disease-causing genes or edit pathogenic mutations (4).Originally discovered in bacteria as part of a natural self-defensemechanism, the Cas9 nuclease—guided by a short guide RNA(sgRNA)—generates double-stranded breaks (DSB) at targeted genomic loci(5).

However, to date, the application of gene editing to neurologic diseaseshas been limited (6). For instance, CRISPR/Cas9 has been used incell-based models to edit triplet-repeat expansions of Huntington's andFragile X syndrome (7, 8). Besides significant technical caveats such aslow editing efficiency and limited in vivo validation (6), suchcanonical approaches would only be applicable to the small fraction ofcases that are inherited (i.e. <10% of AD, Parkinson's, ALS); with adifferent approach required for each gene. Moreover, the feasibility ofCRISPR/Cas9 as a therapeutic possibility in AD has not been reported.

Needed in the art of Alzheimer's disease treatment is an improved methodof using gene editing methods to treat or prevent the disease.

SUMMARY OF THE INVENTION

In a first aspect, provided herein is a method of treating or preventingAlzheimer's disease (AD) caused by formation of amyloid plaques composedof amyloid beta (Aβ) peptides, wherein the method comprises the steps of(a) obtaining a gene-editing construct specific for the amyloidprecursor protein (APP), wherein the construct facilitates truncation ofthe APP C-terminus when combined with a Cas9 nuclease, and (b)delivering the construct and a construct encoding the Cas9 nuclease to apatient in need of AD therapy, wherein the APP molecule is truncated andproduction of Aβ peptides is decreased in the patient's brain. In someembodiments, the truncation of the APP C-terminus occurs at an APPresidue selected from the group consisting of 659, 670, 676, and 686. Insome embodiments, the gene-editing construct comprises a gRNA sequenceselected from the group consisting of SEQ ID NOs:1-10. In someembodiments, the construct and the nuclease are delivered in acomposition comprising an adeno-associated viral vector and ananocarrier delivery vehicle. In some embodiments, the composition isdelivered intravenously or intrathecally.

In a second aspect, provided herein is a method of reducing theformation of amyloid plaques in a patient's brain, wherein the plaquescomprise amyloid beta (Aβ) peptides, the method comprises the steps of(a) obtaining a gene-editing construct specific for the amyloidprecursor protein (APP), wherein the construct facilitates truncation ofthe APP C-terminus when combined with a Cas9 nuclease, and (b)delivering the construct and nuclease to a patient in need of ADtherapy, wherein the APP molecule is truncated and production of Aβpeptides is decreased in the patient's brain. In some embodiments, thetruncation of the APP C-terminus occurs at an APP residue selected fromthe group consisting of 659, 670, 676, and 686. In some embodiments, thegene-editing construct comprises a gRNA sequence selected from the groupconsisting of SEQ ID NO:1-10. In some embodiments, the construct and thenuclease are delivered in a composition comprising an adeno-associatedviral vector and a nanocarrier delivery vehicle. In some embodiments,the composition is delivered intravenously or intrathecally.

In a third aspect, provided herein is a genetic construct comprising, asequence encoding for a Cas9 nuclease and a sequence encoding a gRNAspecific to amyloid precursor protein (APP). In some embodiments, theconstruct is packaged in a viral vector selected from the groupconsisting of a lentiviral vector and an adeno-associated viral (AAV)vector. In some embodiments, the construct further comprises at leastone neuron specific promoter. In some embodiments, the neuron specificpromoter is selected from the group consisting of human synapsin 1(hSyn1) promoter, and mouse Mecp2 promoter (pMecp2). In someembodiments, the construct further comprises an RNA Pol III promoter. Insome embodiments, the RNA Pol III promoter is a U6 promoter. In someembodiments, the sequence of the gRNA is selected from the groupconsisting of SEQ ID NOs:1-10. In some embodiments, the sequence of theCas9 nuclease consists of SEQ ID NO:15. In some embodiments, theconstruct comprises the sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, or SEQ ID NO:20. In some embodiments, the sequence encoding for aCas9 nuclease in packaged on a first AAV vector and the sequenceencoding a gRNA specific to amyloid precursor protein (APP) is packagedon a second AAV vector.

In a forth aspect, provided herein is a kit for reducing the formationof amyloid plaques in a patient's brain, the kit comprising a firstviral vector encoding a gRNA selected from the group consisting of SEQID NOs:1-10 and a second viral vector encoding a Cas9 nuclease. In someembodiments, the viral vector is selected from the group consisting of alentiviral vector and an adeno-associated viral (AAV) vector. In someembodiments, the first or second viral vector further comprises at leastone neuron specific promoter. In some embodiments, the neuron specificpromoter is selected from the group consisting of human synapsin 1(hSyn1) promoter, and mouse Mecp2 promoter (pMecp2). In someembodiments, the first or second viral vector further comprises an RNAPol III promoter. In some embodiments, the RNA Pol III promoter is a U6promoter. In some embodiments, the kit comprises a viral vector encodingboth a gRNA selected from the group consisting of SEQ ID NOs:1-10 and aCas9 nuclease.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1F show manipulation of the amyloid pathway by CRISPR/Cas9editing. (FIG. 1A) Schematic and C-terminal sequence of mouse APPshowing PAM sites (yellow) and genomic targets for the three APP-sgRNAs(APP-659 sgRNA used henceforth and referred to as ‘APP-sgRNA’—see text).Note that the C-terminal antibody Y188 recognizes the last 20 aminoacids of APP. (FIG. 1B) Neuro2A cells were transfected with APP-sgRNAand Cas9 (or Cas9 only), and immunostained with the Y188 antibody (after5 days; mCherry labels transfected cells). Note decreased APP (Y188)fluorescence, indicating APP editing (quantified on right, mean±SEM of39 cells from two independent experiments per condition, p<0.0001).(FIGS. 1C-1D) Neuro2A cells were transduced by lentiviral vectorscarrying APP-sgRNA and Cas9 (or non-targeting control-sgRNA/Cas9 ascontrol) and immunoblotted with Y188 and 22C11 antibodies (latterrecognizes APP N-terminus). A gamma secretase inhibitor (GSI) was addedto allow detection of accumulated APP CTF's (see methods, GAPDH used asloading controls). Note attenuated signal with the Y188 antibody inAPP-sgRNA treated samples, but no change in 22C11 signal. Blotsquantified in (d), mean±SEM of six independent experiments, p<0.0001.(FIG. 1E) Time course of APP-editing in neuro2a cells. Cells weretransfected with a vector carrying APP-sgRNA and Cas9, and APP-CTFs wereanalyzed by Western blotting (in the presence of GSI). (FIG. 1F) Deepsequencing of APP C-terminus in neuro2A cells. Top: Frequency ofbase-pair matches between gRNA-edited and WT mouse sequence. Redunderline marks the sgRNA target sequence and arrowhead denotespredicted cut-site. Note extensive mismatch around predicted cut-site,indicating robust editing. Bottom: Major mutated APP loci resulting fromsgRNA-editing, and their frequencies.

FIGS. 2A-2H show gene editing of APP C-terminus and effects on APPprocessing in human cells. (FIG. 2A) Comparison of mouse and humanAPP-sgRNA targeting sequences (red arrowheads indicate differences;yellow bar denotes the PAM site). (FIG. 2B) Human iPSC-derived NPCs weretransduced by lentiviral vectors carrying APP-sgRNA and Cas9 (ornon-targeting control-sgRNA/Cas9 as control) and differentiated intoneurons. After 3 weeks of differentiation, cells were immunostained withthe Y188 and Tuj 1 (tubulin) antibodies. Note decreased APP (Y188)fluorescence, indicating APP editing. (FIG. 2C) The iPSC-derived neuronsabove (or isogenic APPV717I London-mutant knock-in iPSC-neurons) weretransduced and differentiated as above and immunoblotted with C- andN-terminus antibodies (GSI was added to allow detection of accumulatedAPP CTFs). Note attenuation of APP signal with Y188 after APP-sgRNAtreatment in both wild type and isogenic APP London iPSCs (quantified onright, mean±SEM of three independent experiments, ** p<0.01, ***p<0.001,****p<0.0001). (FIG. 2D) Media from the iPSC-derived neurons above wasimmunoblotted for secreted sAPPa (6E10 antibody). Note increased sAPPain sgRNA-treated samples, indicating upregulation of thenon-amyloidogenic pathway. (FIG. 2E) ELISA of media from iPSC derivedneurons. Note decreased Aβ in the sgRNA-treated samples (mean±SEM ofthree independent experiments, ** p<0.01, ***p<0.001, ****p<0.0001).(FIG. 2F) Deep sequencing of APP C-terminus in human ESCs. Red underlinemarks the sgRNA target sequence and arrowhead denotes predictedcut-site. Note extensive mismatch around predicted cut-site, indicatingrobust editing. (FIG. 2G) Major mutated APP-loci resulting from CRISPRediting, and their frequencies. (FIG. 2H) Predicted APP translationalproducts (post-editing) for the major mutant alleles observed in deepsequencing. Note that after editing, APP is translated up-to amino acid659 (red arrowheads; similar results were seen in HEK cells, see FIG.6E).

FIGS. 3A-3G show the effect of APP C-terminus editing on neuronalphysiology. (FIG. 3A) AAV9-sgRNA and AAV9-Cas9 expression vectors. Notethat the sgRNA vector co-expresses GFP and the Cas9 is tagged to HA, foridentification of transduced neurons. (FIG. 3B) Cultured hippocampalneurons were transduced with AAV9s carrying APP-sgRNA/Cas9 (or Cas9only) and immunoblotted with the Y188 and 22C11 antibodies (in thepresence of GSI). Note attenuation of CTFs by the APP-sgRNA. (FIG. 3C)Neurons were transfected (at the time of plating) with a vectorexpressing APP-sgRNA and Cas9. Neuritic/axon outgrowth was analyzedafter 5-6 days. Neurons were transfected or infected at DIV7 with APPCRISPR, and synapse structure/function was analyzed after 14-17 days.(FIG. 3D) Top: Representative images of neurons transfected with theAPP-sgRNA/Cas9 (or Cas9 alone). Bottom: Axon length and number ofneurites/branches in the APP-sgRNA/Cas9 (or Cas9 alone) groups; notethat there was no significant difference (mean±SEM; axon length: 30cells for Cas9 only and 27 cells for moAPP-sgRNA from two independentexperiments, p=0.2462; neurite number: 35 cells for Cas9 only and 31cells for moAPP-sgRNA from two independent experiments, p=0.2289; branchnumber: 27 cells for both conditions from two independent experiments,p=0.6008). (FIG. 3E) Neurons were infected with AAV9 viruses carryingAPP-sgRNA/Cas9 (or Cas9 only as controls), and fixed/stained with thepresynaptic marker VAMP2. Note that the presynaptic density (VAMP2puncta) was similar in both groups (quantified on right, mean±SEM ofVAMP2 staining along 27 dendrites for Cas9 only and 25 dendrites formoAPP-sgRNA from two independent experiments, p=0.3132). (FIG. 3F)Neurons were transfected with APP-sgRNA/Cas9 (or Cas9 only as controls).Spine density in the APP-sgRNA/Cas9 (or Cas9 only) groups was alsosimilar, quantified on right (mean±SEM of 18 dendrites for Cas9 only and16 dendrites for moAPP-sgRNA from two independent experiments,p=0.7456). (FIG. 3G) Miniature excitatory postsynaptic currents (mEPSC)were recorded from neurons infected with AAV9-APP-sgRNA/Cas9 orAAV9-Cas9 alone. Top: Representative mEPSC traces in control andAPP-sgRNA transduced neurons. Corresponding alignments of mEPSCs withaverage (white traces) are shown on right. Bottom: Cumulative histogramsof mEPSC amplitude, 20-80% rise-time and inter-event interval inAPP-sgRNA/Cas9 and the Cas9-only infected neurons (note no significantdifferences).

FIGS. 4A-4G show gene editing of APP C-terminus in vivo. (FIG. 4A)AAV9-sgRNA and AAV9-Cas9 were stereotactically co-injected into dentategyrus of 8-week old mouse brains (bottom). Two weeks after viraldelivery, brains were perfused, fixed, and immunostained with anti-GFP,anti-HA and anti-APP(Y188) antibodies. (FIG. 4B) Co-expression ofAAV9-sgRNA-GFP and AAV9-HA-Cas9 in the dentate gyrus. Note that majorityof neurons are positive for both GFP and HA (˜87% of the cells werepositive for both; sampling from 3 brains). (FIGS. 4C-4D) Coronalsection of a mouse hippocampi injected on one side (marked by arrow)with the AAV viruses as described above. Note attenuated Y188 stainingof neurons on the injected side, indicating APP-editing. The image ofmouse hippocampus injected with Cas9 only is not shown. Fluorescencequantified in (d), mean±SEM, data from three brains. One-way ANOVA:p<0.0001. Tukey's multiple comparisons: p=0.4525 (Un-injected vs Cas9only); p<0.0001 (Un-injected vs APP-sgRNA); p<0.0001 (Cas9 only vsAPP-sgRNA). (FIG. 4E) Intracerebroventrical injection of the AAV9viruses into P0 pups. Note widespread delivery of gRNA into brain, asevident by GFP fluorescence. (FIG. 4F) Brain sections from above wereimmunostained with the Y188 antibody. Note attenuated Y188 staining inthe APP-sgRNA/Cas9 transduced sample, suggesting APP-editing. (FIG. 4G)Western blots of the brains from (e). Note decreased expression of CTFsin the APP-sgRNA/Cas9 transduced brains; blots quantified on right(mean±SEM of three independent experiments, **p<0.01).

FIGS. 5A-5E show mechanistic details of CRISPR-guided APP editing. (FIG.5A) APP/BACE-1 interaction—as evaluated by fluorescence complementationin cultured hippocampal neurons—was attenuated in neurons transfectedwith an APP C-terminus truncation mimicking the post-editedtranslational product (APP659:VN; quantified below, mean±SEM of 12 cellsfor APP(WT) and 13 cells for APP(659) from two independent experiments,p<0.0001). (FIG. 5B) APP β-cleavage is also attenuated in cellstransfected with APP659. HEK cells were co-transfected with APPWT (orAPP659) tagged to VN, and BACE-1:VC; and immunoblotted with the 6E10antibody. Note decreased β-CTFs in cells carrying the truncated APPplasmid. (FIG. 5C) Schematic showing the CRISPR-edited C-terminusportion of APP. Note that the threonine at 668 position, and theendocytic YENPTY motif (dashed boxes) are thought to play roles in Aβproduction (see text). (FIG. 5D) APP/BACE-1 interaction—as evaluated byfluorescence complementation in cultured hippocampal neurons—was mostmarkedly attenuated in neurons transfected with mutant YENPTY (mean±SEMof 32 cells for APP(WT), 37 cells for APP(T668A), 45 cells forAPP(YENPTY) and 49 cells for APP(T668A+YENPTY) from two independentexperiments). One-way ANOVA: p<0.0001. Tukey's multiple comparisons:p=0.0022 (APP vs APP^(T668A))^(; p)<0.0001 (APP vs APP^(YENPTY));p<0.0001 (APP vs APP^(T668A+YENPTY)); <0.0001 (APP^(T668A) vsAPP^(YENPTY)); p<0.0001 (APP^(T668A) vs APP^(T668A+YENPTY)); p=0.7568(APP^(YENPTY) vs APP^(T668A+YENPTY)). (FIG. 5E) Strategy of APPinternalization assay. Neuro 2a cells are transfected with APP:GFP orAPP659:GFP. After incubation with anti N-terminal APP antibody (22C11)for 10 min, the cells were fixed and stained with secondary antibody tovisualize the cell surface and internalized APP. Note the cell surfaceaccumulation and decreased internalization of APP659 (mean±SEM of 21cells from two independent experiments, p<0.0001).

FIGS. 6A-6E show the choice of CRISPR editing site at APP C-terminus.(FIG. 6A) Strategy to integrate APP:VN and BACE-1:VC into the H4 genomeand generation of a stable cell line expressing single copies of the twoproteins (see results and methods for details). (FIG. 6B) APP and BACE-1expression in the H4^(single copy) cell line. Note negligible expressionof endogenous proteins in native H4 cells. (FIG. 6C) TheH4^(single copy) cell line was transduced with lentiviral vectorscarrying non-targeting control-sgRNA/Cas9 or various human APPC-terminus targeting sgRNAs/Cas9 (see Table 5 for targeting sequences).The APP/BACE-1 Venus complementation was visualized by fluorescencemicroscopy. Note attenuation of complementation, indicating editing bythe APP-sgRNAs (quantified on right, mean±SEM of three independentexperiments). One-way ANOVA: p<0.0001. Tukey's multiple comparisons:p<0.0001 (control-sgRNA vs APP659-sgRNA); p<0.0001 (control-sgRNA vsAPP670-sgRNA); p<0.0001 (control-sgRNA vs APP676-sgRNA); p=0.0064(APP659-sgRNA vs APP670-sgRNA); p=0.0015 (APP659-sgRNA vs APP676-sgRNA);p=0.6207 (APP670-sgRNA vs APP676-sgRNA). (FIG. 6D) ELISA of media fromthe H4^(single copy) cell line (treated as above). Note decreased Aβ inthe APP-sgRNAs treated samples (mean±SEM of three independentexperiments). One-way ANOVA for Aβ 40 and 42: p<0.0001. Tukey's multiplecomparisons for Aβ 40: p<0.0001 (control-sgRNA vs APP659-sgRNA;control-sgRNA vs APP670-sgRNA; control-sgRNA vs APP676-sgRNA); p=0.0331(APP659-sgRNA vs APP670-sgRNA); p=0.0071 (APP659-sgRNA vs APP676-sgRNA);p=0.6673 (APP670-sgRNA vs APP676-sgRNA). Tukey's multiple comparisonsfor Aβ 42: p<0.0001 (control-sgRNA vs APP659-sgRNA; control-sgRNA vsAPP670-sgRNA; control-sgRNA vs APP676-sgRNA); p=0.0068 (APP659-sgRNA vsAPP670-sgRNA); p=0.0221 (APP659-sgRNA vs APP676-sgRNA); p=0.8079(APP670-sgRNA vs APP676-sgRNA). (FIG. 6E) HEK cells were transduced bylentiviral vectors carrying APP-sgRNAs and Cas9 (or non-targetingcontrol-sgRNA/Cas9 as control), and APP C-terminus was sequenced. Left:Deep sequencing of APP659-sgRNA treated cells, and Sanger sequencingfollowed by ICE analyses for APP670-sgRNA and APP676-sgRNA treatedcells. Red underlines mark the sgRNA-targeting sequences and arrowheadsdenote predicted cut-sites. Right: Predicted APP translational productsafter CRISPR/Cas9 editing in human HEK cells for the major mutantalleles observed in sequencing analyses. Red arrowheads indicate theamino acids where APP genes were translated up to after editing.

FIGS. 7A-7D show evaluation of CRISPR editing by immunoblotting in mouseNeuro2a cells. (FIG. 7A) Neuro2a cells were co-transfected with a sgRNAthat knocked out the entire APP gene and Cas9 (see Table 5 for APPtargeting sequence), and immunostained with APP N-terminal andC-terminal antibodies (after 5 days in culture). Note attenuation ofstaining for both Y188 and 22C11. (FIG. 7B) Neuro2a cells weretransfected with various APP C-terminus targeting sgRNAs (ornon-targeting control-sgRNA), and immunostained with APP N-terminal andC-terminal antibodies (after 5 days in culture in the presence of GSI).Note attenuation of staining by Y188 but not 22C11, indicating selectiveediting of the APP C-terminus. (FIG. 7C) Neuro2A cells were transducedby lentiviral vectors carrying APP-sgRNA and Cas9 (or non-targetingcontrol-sgRNA/Cas9 as control) and immunoblotted with the APP antibodiesCT20 and M3.2 (CT20 recognizes last 20 aa; M3.2 recognizes anextracellular domain located upstream of the CRISPR/Cas9 targetingsite). A GSI was added to allow detection of accumulated APP CTF's. Noteattenuated signal with CT20- but not M3.2-antibody, indicating selectiveediting of the APP C-terminus. (FIG. 7D) Post-editing translationalproducts in mouse (neuro 2a) cells. Note effective truncation of APP ataa 659.

FIGS. 8A-8G show APP C-terminus editing by CRISPR/Cas9. (FIG. 8A) HEKcells were transfected with human-specific APP-sgRNA and Cas9 (or Cas9only), and immunostained with the Y188 antibody (after 5 days inculture). Note attenuation of staining, quantified on right (mean±SEM of25 cells for Cas9 only and 43 cells for huAPP-sgRNA from two independentexperiments, p<0.0001). (FIG. 8B) HEK cells were transduced bylentiviral vectors carrying APP-sgRNA and Cas9 (or non-targetingcontrol-sgRNA/Cas9 as control) and immunoblotted with the Y188 and 22C11antibodies (in the presence of GSI). Note attenuation of APP-CTFs inAPP-sgRNA treated cells, indicating CRISPR-editing (mean±SEM of threeindependent experiments, p<0.0001). (FIG. 8C) HEK cells above wereimmunoblotted with CT20 and 2E9 antibodies (CT20 recognizes last 20 aa;2E9 recognizes APP extracellular domain upstream of the CRISPR/Cas9targeting site). Note attenuated signal with CT20- but not 2E9-antibody,indicating selective editing of the APP C-terminus. (FIGS. 8D-8E) HumanESCs were transduced by lentiviral vectors carrying human APP-sgRNA/Cas9(or non-targeting sgRNA/Cas9). Samples were immunostained with the Y188antibody (d) or immunoblotted with the Y188 and 22C11 antibodies (e).Note attenuation of APP-CTFs in sgRNA-transduced group (forimmunostaining, mean±SEM of 17 colonies for control-sgRNA and 20colonies for huAPP-sgRNA from two independent experiments, p<0.0001; forwestern blotting, mean±SEM of three independent experiments, p=0.001 fortotal APP and p<0.0001 for CTFs). (FIG. 8F) Media from iPSC derivedneurons were immunoblotted for extracellular sAPPβ (in the absence ofGSI). Note decrease in APP β-cleavage in the APP-sgRNA treated samples.(FIG. 8G) Media from H4^(single-copy) cells were immunoblotted forextracellular sAPPα with 6E10 antibody and sAPPβ (in the absence ofGSI). Note enhanced APP α-cleavage and attenuated APP β-cleavage in theAPP-sgRNA treated samples.

FIGS. 9A-9C show gene editing by APP-sgRNA likely does not influence APPγ-cleavage. (FIG. 9A) Strategy to evaluate γ-cleavage of post-editedAPP. Neuro2a cells were transfected with either full length (FL) C99, orC99 truncated at aa 659 (to mimic the post-editing translationalproduct; all constructs were GFP-tagged to confirm expression).γ-cleavage of the FL and 659 C99 was evaluated by western blotting (notethat neuro2a cells have all components of the γ-secretase complex).(FIG. 9B) Schematic showing expected C99-cleavage patterns. Note thatupon γ-cleavage, both C99-fragments will be further truncated. However,if the ‘CRISPR-mimic’ (659) fragment did not undergo γ-cleavage, thistruncation would not occur. (FIG. 9C) Western blotting of the cells from(a) indicates that both C99 fragments (FL and 659) undergo γ-cleavage—asindicated by the shift upon inhibiting γ-cleavage by GSI. These datasuggest that gene editing by the APP-gRNA likely does not affect APPγ-cleavage, and that the effects seen on the amyloid pathway are likelydue to modulation of APP-β-cleavage.

FIGS. 10A-10G show off target analyses of APP-sgRNA. (FIG. 10A)Computationally predicted top five off-target (OT) sites in the genome,that can be potentially targeted by the mouse and human APP-sgRNAs(mismatched nucleotides in the targeting sequence are marked in red).Genomic locations corresponding to the sequences is shown on the rightcolumn (note most are in non-coding regions). (FIG. 10B) Strategy of T7endonuclease digestion assay to detect genome-editing events. GenomicDNA was PCR amplified with primers bracketing the modified locus. PCRproducts were then rehybridized, yielding three possible structures.Duplexes containing a mismatch were digested by T7 endonuclease I. DNAgel analysis was used to calculate targeting efficiency. Note digestedfragments in the gel indicates cleavage. (FIG. 10C) Gene edits at theAPP locus by the APP-sgRNA, as seen by T7 endonuclease digestion. Notetwo digested fragments were recognized after T7 endonuclease digestion.(FIGS. 10D-10E) T7 endonuclease assays of potential off-target sites(mouse and human). No digested fragments are seen, indicating that thesgRNAs do not generate detectable gene edits at these sites. (FIG. 10F)Comparison of APLP1 and 2 sequences with APP at the sgRNA targetingsite. Asterisks mark conserved nucleotide sequences, and the PAM sitesare underlined. Nucleotide mis-matches are highlighted in yellow. Noteextensive mis-match of the mouse and human sequences at the sgRNAtargeting site. (FIG. 10G) Left: Off-target TIDE analysis of APP familymembers APLP1 and 2 in mouse (neuro 2a) and human (HEK) cell linesfollowing lentiviral integration of Cas9 using TIDE. No modificationswere detected below the TIDE limit of detection (dotted line) in eitherof the populations, indicating that the APP-gRNA was unable to edit APLP1/2. Right: TIDE analysis of APLP1 and 2 loci in mouse and human celllines. Neither of the populations had significant editing at either ofthe two loci, and all sequences had a near perfect correlation to themodel.

FIGS. 11A-11C show trafficking of vesicles carrying APP(WT) or APP(659).(FIG. 11A) Cultured hippocampal neurons were transfected withAPP(WT):GFP or APP(659):GFP, and kinetics of APP particles were imagedlive in axons and dendrites. (FIG. 11B) Representative kymographs andquantification of APP kinetics in axons. Note that there was no changein frequency of transport, and only a modest reduction in run-length andvelocity. Error bars, mean±SEM of 325 APP(WT):GFP and 310 APP(659):GFPvesicles in 10-12 neurons from two independent experiments. Frequency:p=0.4635 (APP_antero vs APP659_antero); p=0.6650 (APP_retro vsAPP659_retro); p=0.7420 (APP_stat vs APP659 stat). Velocity: P<0.0001(APP_antero vs APP659_antero); p=0.9419 (APP_retro vs APP659_retro). Runlength: p<0.0001 (APP_antero vs APP659_antero); p=0.2433 (APP_retro vsAPP659_retro). (FIG. 11C) Representative kymographs and quantificationof APP kinetics in dendrites. Error bars, mean±SEM of 130 APP(WT):GFPand 115 APP(659):GFP particles in 10-12 neurons from two independentexperiments. Frequency: p=0.3245 (APP_antero vs APP659_antero); p=0.5438(APP_retro vs APP659_retro); p=0.2394 (APP_stat vs APP659_stat).Velocity: p=0.0120 (APP_antero vs APP659_antero); p=0.6248 (APP_retro vsAPP659_retro). Run length: p=0.1352 (APP_antero vs APP659_antero);p=0.4284 (APP_retro vs APP659_retro).

FIGS. 12A-12C show internalization of APP-659-GG (most commonpost-editing translational product). (FIGS. 12A-12B) Neuro2a cells wereco-transfected with untagged APP-659-GG and mCherry (or untagged WT APPand mCherry as control). After incubation with anti N-terminal APPantibody (22C11) for 10 min, the cells were fixed and stained withsecondary antibody to visualize surface and internalized APP (mCherrylabels transfected cells). Note accumulation of APP-659-GG on the cellsurface, along with decreased internalization; quantified in FIG. 12B.Mean±SEM of 25 cells for APP(WT) and 26 cells for APP-659-GG from twoindependent experiments, p<0.0001. (FIG. 12C) Expression levels ofexogenous APP constructs. Note that WT and APP-659-GG were expressed atsimilar levels in the Neuro2a cells above.

FIG. 13 shows the sequence of human APP (nucleotide sequence SEQ IDNO:11, amino acid sequence SEQ ID NO:12) and mouse APP (nucleotidesequence SEQ ID NO:13, amino acid sequence SEQ ID NO:14) along with thecorresponding sequences of the gRNA used in select gene editingembodiments described herein.

FIG. 14 shows the sequence of the Cas9 nuclease gene sequence.

FIG. 15 shows the sequence and vector map of an exemplary vector (SEQ IDNO:17) for APP truncation at amino acid 659. The vector includes thegRNA sequence (lowercase italics) and the Cas9 nuclease sequence.

FIG. 16 shows the sequence and vector map of an exemplary Cas9 vector(SEQ ID NO:18).

FIG. 17 shows the sequence and vector map of an exemplary APP sgRNAvector (SEQ ID NO:19).

FIG. 18 shows the sequence and vector map of an exemplary lentiviral APPsgRNA vector (SEQ ID NO:20).

INCORPORATION BY REFERENCE

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

DETAILED DESCRIPTION OF THE INVENTION In General

Gene-editing methods, such as CRISPR/Cas9 guided gene-editing, holdpromise as a therapeutic tool. However, few studies have applied thetechnology to neurodegenerative diseases. Moreover, the conventionalapproach of mutation-correction is limited in scope to inheriteddiseases which are a small fraction of neurodegenerative disease cases.The present invention introduces a strategy to edit endogenous amyloidprecursor protein (APP) at the extreme C-terminus and selectivelyattenuate the amyloidogenic pathway—a key pathologic cascade inAlzheimer's disease (AD). In the method of the present invention, theAPP N-terminus remains intact and protective α-cleavage is up-regulated.

The Examples below demonstrate that robust APP-editing is demonstratedin cell lines, human stem cells, cultured neurons, and in mouse brains.Physiologic parameters remain unaffected. Without being bound by anyparticular theory, the present invention works by restricting thephysical interaction of APP and BACE-1, said interaction being therate-limiting step in amyloid-β (Aβ) production. The Examples belowdelineate underlying mechanisms that abrogate APP/BACE-1 interaction inthis setting. The invention offers an innovative ‘cut and silence’gene-editing strategy that could be a new therapeutic paradigm for AD.

CRISPR/Cas9 works by inducing sequence-specific double-stranded breaks(DSBs) in DNA. After such breaks, the cell undergoes an error-pronerepair process called non-homologous end joining, leading to adisruption in the translational reading frame, often resulting inframeshift mutations and premature stop codons. For the system to work,at least two components must be introduced in cells: a Cas9 nuclease anda guide RNA. Described herein are CRISPR/Cas9 constructs suitable fortruncation of the APP protein and disruption of amyloid-β production.

Constructs of the Present Invention

In a first aspect, the present invention provides a construct for CRISPRmediated cleavage of the APP gene. The constructs of the presentinvention include a nucleotide sequence encoding a Cas9 nuclease and aguide RNA (gRNA). In some embodiments the sequence encoding the Cas9nuclease and the gRNA are included on a single vector construct. In someembodiments the sequence encoding the Cas9 nuclease is included in avector construct separate from a vector construct encoding for the gRNA.Additionally, the construct may include a promoter, a poly(A) tail, anoptional reporter element, and an optional selection marker such as anampicillin selection marker.

As used herein “Cas9 nuclease” refers to the RNA-guided DNA endonucleaseenzyme associated with the CRISPR adaptive immunity system inStreptococcus pyogenes and other bacteria. The Cas9 nuclease includestwo nuclease domains, a RuvC-like nuclease domain located at the aminoterminus, and a HNH-like nuclease domain. In some embodiments, thesequence of the Cas9 nuclease is the sequence included in FIG. 14 (SEQID NO:15).

In some embodiments, the Cas nuclease is expressed under the control ofa neuron specific promoter or ubiquitous promoter. The neuron specificpromoter may be any neuron specific promoter known in the art (see forexample, Swiech L et al., In vivo interrogation of gene function in themammalian brain using CRISPR-Cas9. Nature Biotechnology 2015 January;33(1): 102-6). In some embodiments the neuron specific promoter is thehuman synapsin 1 (hSyn1) promoter. In some embodiments the neuronspecific promoter is the mouse Mecp2 promoter (pMecp2). In someembodiments the ubiquitous promoter is the chicken β-actin promoter. Insome embodiments, the ubiquitous promoter is an EFS promoter.

In one embodiment of the present invention, the construct is specificfor the extreme C-terminus of the APP gene. By “APP gene” or “amyloidprecursor protein”, we mean to include the human APP gene as disclosedin Hendricks et al (Hendriks L et al. Presenile dementia and cerebralhaemorrhage linked to a mutation at codon 692 of the beta-amyloidprecursor protein gene. Nature Genetics 1992 June; 1(3): 218-21) andrecited herein as SEQ ID NO:11. The amino acid sequence of the APP geneis recited as SEQ ID NO:12.

As used herein “extreme C-terminus,” refers of a portion of theC-terminus of the APP protein which, when absent, is sufficient todisrupt the interaction between APP and BACE. The truncated APP lackingthe extreme C-terminus will still include its native N-terminus, thetransmembrane domain and the residual C-terminal region. Typically, theextreme C-terminus of the APP protein will mean 8 or more amino acids atthe C-terminus of the APP protein. This may be accomplished byCRISPR/Cas9 mediated cleavage of the APP gene such that the expressedAPP protein is truncated to a length selected from the group consistingof 659, 670, 676, or 686, relative to SEQ ID NO:12 (human) or SEQ IDNO:14 (mouse). In some embodiments, the APP gene is cleaved following anucleotide selected from the group consisting of 1978, 2009, 2010, 2029,and 2058 relative to SEQ ID NO:11 (human) or SEQ ID NO:14 (mouse). Alist of these cleavage sites is included in the table below.

As used herein “guide RNA (gRNA)” refers to the 20 nucleotide targetsequence which directs Cas9 mediated cleavage within the APP gene. ThegRNA will be encoded on a synthetic RNA construct which additionallyincludes the tracrRNA sequence. While the gRNA sequence is variable andwill be specific for the cleavage site of interest, the tracrRNA is thesame for all gRNA sequences used. The tracrRNA sequence is SEQ ID NO:16The gRNA described herein are specific for the truncation of theC-terminal segment of APP. Suitable target sequences within the APP genefor design of gRNA sequences are recited below, which includes thesequence of the gRNA.

tracrRNA (SEQ ID NO:16):  5′-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3

TABLE 1  Human APP (full length 695 aa) Cas9 Length cleavage of sgRNA SEQ Position between trunca- sgRNA sequence  ID relative nucleo- tedname (5′-3′) NO: to APP tides protein sgRNA 1 atccattcat 1 1962-19811978/1979 659 aa catggtgtgg sgRNA 2 tggacaggtg 2 2007-2026 2009/2010670 aa gcgctcctct sgRNA 3 ttggacaggt 3 2008-2027 2010/2011 670 aaggcgctcctc sgRNA 4 gtagccgttc 4 2027-2046 2029/2030 676 aa tgctgcatctsgRNA 5 tgctcaaaga 5 2056-2075 2058/2059 686 aa acttgtaggt

TABLE 2  Mouse APP (full length 695 aa) Cas9 Length Position cleavage ofsgRNA  SEQ relative between trunca- sgRNA sequence ID to nucleo- tedname (5′-3′) NO: APP tides protein sgRNA atccatccat  6 1962-19811978/1979 659 aa 1 catggcgtgg sgRNA tggagagatg  7 2007-2026 2009/2010670 aa 2 gcgctcctct sgRNA ttggagagat  8 2008-2027 2010/2011 670 aa 3ggcgctcctc sgRNA atatccgttc  9 2027-2046 2029/2030 676 aa 4 tgctgcatctsgRNA tgctcaaaga 10 2056-2075 2058/2059 686 aa 5 acttgtaagt

Cleavage of the APP gene will occur between the 3rd and 4th nucleotidesfrom the PAM site associated with the target sequence in the APP gene.For the sgRNA 1, the PAM site is on the sense strand of the APP gene,the sgRNA of SEQ ID NOs:1 and 6 are complementary to the antisensestrand of the APP gene, and the cleavage will occur between nucleotides1978 and 1979 relative to SEQ ID NO:11 (human) or SEQ ID NO:14 (mouse).For sgRNA 2, 3, 4 and 5, the PAM site is on the antisense strand of theAPP gene, the sgRNA of SEQ ID NOs:2-5 and 7-10 are complementary to thesense strand of the APP gene, and the cleavage site is betweennucleotides 2009 and 2010 for sgRNA 2, between nucleotides 2010 and 2011for sgRNA 3, between nucleotides 2029 and 2030 for sgRNA 4 and betweennucleotides 2058 and 2059 for sgRNA 5, relative to SEQ ID NO:11 (human)or SEQ ID NO:14 (mouse).

In some embodiments, the gRNA or tracrRNA is modified by any means knownin the art. Common methods for gRNA or tracrRNA modification includechemical modifications or modifications to axillary sequences appendedto the RNA to increase efficiency known in the art.

In some embodiments, the gRNA is expressed under the control of an RNAPol III promoter. Examples of RNA Pol III promoters include, but are notlimited to, U6 and H1 promoters. A promoter, generally, is a region ofnucleic acid that initiates transcription of a nucleic acid encoding aproduct. A promoter may be located upstream (e.g., 0 bp to −100 bp, −30bp, −75 bp, or −90 bp) from the transcriptional start site of a nucleicacid encoding a product, or a transcription start site may be locatedwithin a promoter. A promoter may have a length of 100-1000 nucleotidebase pairs, or 50-2000 nucleotide base pairs. In some embodiments,promoters have a length of at least 2 kilobases (e.g., 2-5 kb, 2-4 kb,or 2-3 kb).

In some embodiments, the construct comprises an optional reporterelement. The reporter element may be any reporter known in the artincluding, but not limited to, mCherry, green fluorescent protein, andhuman influenza hemagglutinin (HA).

In some embodiments, the constructs are packaged in a vector suitablefor delivery into a mammalian cell, including but not limited to, anadeno-associated viral (AAV) vector, a lentiviral vector, or a vectorsuitable for transient transfection. Suitable vector backbones are knownand commercially available in the art. For example, see Deverman et al.(Cre-dependent selection yields AAV variants for widespread genetransfer to the adult brain, Nature Biotechnology, 34(2):204-209, 2016)and Chan et al. (Engineered AAVs for efficient noninvasive gene deliveryto the central and peripheral nervous system, Nature Neuroscience,20(8):1172-1179, 2017) which are incorporated herein by reference intheir entirety. In some embodiments, the vector is an AAV vector and thegRNA and Cas9 constructs are encoded on separate vectors. In someembodiments, the vector is a lentiviral vector and the gRNA and Cas9constructs are encoded on a single vector. In some embodiments, thevector is a vector suitable for transient transfection and the gRNA andCas9 constructs are encoded on a single vector. In one embodiment thevector includes the sequence of SEQ ID NO:17. In some embodiment, thegRNA and Cas9 constructs are encoded on separate AAV vectors wherein thegRNA is encoded on a vector comprising SEQ ID NO:19 and the Cas9construct is encoded on a vector comprising SEQ ID NO:18. In someembodiments, the vector is a lentiviral vector and comprises thesequence of SEQ ID NO:20. The vectors of SEQ ID NOs:17-20 are includedin FIGS. 15-18.

In some embodiments, the vectors encoding the constructs describedherein may optionally include a monoclonal antibody tag (e.g., FLAG),one or more origins of replication (e.g., fl ori), one or moreterminator sequences (e.g. bGH), one or more polyadenylation tags (bGHpoly(A)), and one or more inverted terminal repeats (ITR). The vectormay also include one or more selectable markers, such as anantibacterial resistance marker such as an ampicillin selectable marker.A skilled artisan will be familiar with the elements and configurationsnecessary for vector construction to encode the constructs describedherein.

Methods of the Present Invention

The constructs described herein may be formulated with apharmaceutically acceptable carrier for administration to a patient inneed thereof. A pharmaceutically acceptable carrier may be, but is notlimited to, a nanoparticle cage including the one or more vectors of thepresent invention.

To function as therapeutic agents, the constructs described herein aredelivered into neurons in the patient's brain, crossing the blood brainbarrier (BBB). In one embodiment, one would attach or associate theCRISPR components with a delivery system, such as a nanoparticledelivery system. In some embodiments, the constructs are formulatedusing an AAV vector and are delivered intravenously. In someembodiments, the constructs are delivered intrathecally into the spinalfluid of the patient. In some embodiments, the constructs are deliverdirectly into the brain of the patient.

As used herein, the terms “treat” and “treating” refer to therapeuticmeasures, wherein the object is to slow down or alleviate (lessen) anundesired physiological change or pathological disorder resulting fromAlzheimer's disease. For purposes of this invention, treating thedisease, condition, or injury includes, without limitation, alleviatingone or more clinical indications, reducing the severity of one or moreclinical indications of Alzheimer's disease, diminishing the extent ofthe condition, stabilizing the subject's Alzheimer's disease (i.e., notworsening), delay or slowing, halting, or reversing Alzheimer's diseaseand bringing about partial or complete remission Alzheimer's disease.Treating Alzheimer's disease also includes prolonging survival by days,weeks, months, or years as compared to prognosis if treated according tostandard medical practice not incorporating treatment with theconstructs described herein.

Subjects in need of treatment can include those already having ordiagnosed with Alzheimer's disease as well as those prone to, likely todevelop, or suspected of having Alzheimer's disease, such as a subjectwith a genetic predisposition to or family history of Alzheimer'sdisease. Subjects in need to treatment may be those with a familial ADmutation or wild-type patients without a mutation. In some embodiments,a subject in need of treatment may be a subject who had been diagnosedby a positron emission tomography (PET) scan, a blood test or othermeans known in the art to have AD or to be predisposed to AD.Pre-treating or preventing Alzheimer's disease according to a method ofthe present invention includes initiating the administration of atherapeutic (e.g., the APP gRNA and Cas9 constructs described herein) ata time prior to the appearance or existence of the disease or injury, orprior to the exposure of a subject to factors known to induceAlzheimer's disease. Pre-treating the disorder is particularlyapplicable to subjects at risk of having or acquiring the diseaseinjury.

As used herein, the terms “prevent” and “preventing” refer toprophylactic or preventive measures intended to inhibit undesirablephysiological changes or the development of Alzheimer's disease. Inexemplary embodiments, preventing Alzheimer's disease comprisesinitiating the administration of a therapeutic (e.g., the APP gRNA andCas9 constructs described herein) at a time prior to the appearance orexistence of Alzheimer's disease such that the disease, or its symptoms,pathological features, consequences, or adverse effects do not occur. Insuch cases, a method of the invention for preventing Alzheimer's diseasecomprises administering the APP gRNA and Cas9 constructs describedherein to a subject in need thereof prior to the onset or development ofAlzheimer's disease in a patient at risk for Alzheimer's disease such asa patient with a genetic risk factor or a patient with a family historyof Alzheimer's disease.

As used herein, the terms “subject” or “patient” are usedinterchangeably and can encompass a human or mouse. As used herein, thephrase “in need thereof” indicates the state of the subject, whereintherapeutic or preventative measures are desirable. Such a state caninclude, but is not limited to, subjects having Alzheimer's disease or apathological symptom or feature associated with Alzheimer's disease.

Examples

The embodiment described in this example demonstrates truncation of theC-terminus of the APP protein, attenuation of APP-β-cleavage and Aβproduction, and manipulation of the amyloid pathway using CRISPR/Cas9gene editing.

A common theme in neurodegenerative diseases is that proteins normallypresent in the brain (APP, tau, α-synuclein, TDP-43, etc.) acquire toxicproperties—or trigger pathologic cascades—that ultimately lead tosynaptic loss and neurodegeneration. Our broad idea is to rationallyedit small segments of endogenous proteins known to play key roles inthe progression of disease, with the ultimate goal of attenuating theirpathologic activity. As endogenous proteins expectedly play physiologicroles, it is also important to conserve their normal function, as far aspossible. Here we show conceptual proof of this ‘selective silencing’approach for APP. APP is a single-pass transmembrane protein, cleaved bythe enzymes β- and γ-secretases to ultimately generate Aβ—aneuropathologic hallmark of AD. APP cleavage by the β-secretase BACE-1is the rate limiting step in this ‘amyloidogenic’ pathway.Alternatively, APP is cleaved by α-secretases—the ‘non-amyloidogenic’pathway—that is thought to be neuroprotective because it precludesβ-cleavage of APP (6,7); and studies have highlighted neuroprotectiveeffects of APP-α-cleavage products in vivo (8,9).

We recently developed a Bi-molecular fluorescence complementation (BifC)assay to visualize the physical approximation of APP and BACE-1 inneurons (10). As a control for validation, we found that a C-terminaldeletion also abrogated APP/BACE-1 complementation (10); in line withprevious studies showing that deletions/mutations of the APP C-terminuscan attenuate Aβ production (11-13). Collectively, these observationsoriginally gave us the idea of using CRISPR/Cas9-mediated truncation ofnative APP to attenuate APP-β-cleavage and Aβ production in AD. UsingCRISPR-tools, cell/molecular biology, live imaging, deep sequencing,electrophysiology and in vivo animal studies, here we highlight astrategy to favorably manipulate the amyloid pathway by gene editing.

Results and Discussion

CRISPR/Cas9 editing of APP C-terminus—The CRISPR/Cas9 system consists ofa Cas9 nuclease enzyme that generates double-stranded breaks in DNA, anda custom-designed single guide-RNA (sgRNA) that targets the Cas9 tospecific sites in the host genomic DNA. Typically, the synthetic sgRNAsare complementary to stretches of genomic DNA containing 3-nt PAM(protospacer adjacent motif) and flanking 20-nt sequences. Sincesubsequent repair after DNA-breaks is naturally error-prone, insertionsand deletions (indels) are generated at the cut-sites, leading todisruption of the translational reading frame and effectively truncatedproteins (reviewed in 14). We identified three PAM sites at the APPC-terminus that are conserved in both human and mouse, and synthesizedsgRNAs targeting these regions (FIG. 1A). To compare the editingefficiency of these sgRNAs, we engineered a stable H4 neuroglioma cellline expressing single copies of APP:VN and BACE-1:VC(APP/BACE^(single_copy)), where editing efficiency of a given sgRNAcould be determined as a simple fluorescence on/off readout and theeffect of APP truncation could be assessed by evaluating secreted Aβ(for details, see FIGS. 6A and 6B and methods). The APP-sgRNA predictedto cut human APP at the 659 aa. (amino acid) position was the mostefficient—both in editing APP as well as in attenuating Aβ—and also ledto minimal indels (FIGS. 6C-6E). Accordingly, we used the APP659-sgRNAfor further characterization (henceforth called ‘mo-APP-sgRNA’ or‘hu-APP-sgRNA’ representing mouse and human specific sequences).

The TGG PAM and preceding 20-nt genomic target sequence recognized bythe mo-APP-sgRNA is shown in FIG. 1A (top right); and FIGS. 1B-1F showsgene editing by this sgRNA in mouse cells. Note that upon editing, theY188 antibody—recognizing the last 20 amino acids of APP—would not beable to identify the resultant translational product. Robust editing ofendogenous APP was seen in mouse neuroblastoma cells, as determined byattenuation of immunofluorescence with the Y188 antibody (FIG. 1B), anddecreased Y188-signal in western blots (FIGS. 1C-1D; FIG. 1E showstime-course of editing). Note that the edited APP is recognized byantibodies to the N-terminus, indicating selective editing of theC-terminus by the APP-sgRNA (FIGS. 1C and 1E). However, the N-terminusantibody was unable to detect APP when the entire gene was deleted (FIG.7A). Similar results were obtained with other sgRNAs targeting APPC-terminus and other C- and N-terminus APP antibodies (FIGS. 7B and 7C).Genomic deep-sequencing confirmed efficient editing of mouse APP at theexpected loci, APP-659 (FIG. 1F). Post-editing translational productsshow that the last 36 amino acids are effectively truncated by APP-sgRNA(FIG. 7D). Though the TGG PAM at this site is conserved in both mouseand human APP, and the upstream sgRNA-targeting sequences only differ bytwo nucleotides (FIG. 2A, arrowheads); the mouse APP-sgRNA was unable toedit human APP (not shown). However, a sgRNA specific to the human APPtargeting sequence robustly edited APP in HEK293 (FIGS. 8A-8C), as wellas in human embryonic stem cells (FIGS. 8D-8E). CRISPR editing of APPdid not alter the steady-state levels of holo-APP (note data throughoutwith multiple N-terminus antibodies in various cell lines).

Reciprocal Manipulation of the APP β/α Pathway by CRISPR/Cas9Editing—Next, we examined APP editing in human iPSC-derived neurons. Asshown in FIG. 2B, immunostaining with the Y188 antibody was attenuatedin iPSC-neurons transduced by the hu-APP-sgRNA. To examine effects ofAPP editing in an “AD-like setting”, we also tested the hu-APP-sgRNA ina heterozygous knock-in iPSC line carrying the most common familial ADmutation (APPV717I, also called the ‘London mutation’; see methods fordetails of iPSC line). Both cell-lysates and supernatants were examined,to look for cellular and secreted APP products (see schematic in FIG.2C). Immunoblotting with the Y188 antibody confirmed robust—andC-terminus selective—APP editing in both WT and APP-London iPSC lines(FIG. 2C). Examination of supernatants revealed that interestingly,APP-editing also led to increased sAPPα in both WT and London lines(FIG. 2D); suggesting upregulation of the neuroprotective α-cleavagepathway. ELISAs and western blot showed attenuated secretion of Aβ40/42(FIG. 2E) and sAPPβ (FIG. 8F), confirming inhibition of theamyloidogenic pathway in these neurons. Genomic deep sequencing showedefficient editing of human APP by the sgRNA, with truncation of the last36 amino acids in human embryonic stem cells (FIGS. 2F-2H).

The data from iPSC-neurons suggest that the APP-sgRNA has reciprocaleffects on APP β- and α-cleavage. To validate this in a more controlledsetting, we tested the effects of APP editing in the H4APP/BACE^(single_copy) cell line, where APP-cleavage is tightlyregulated. In line with the data from iPSC-neurons, the hu-APP-sgRNA hadreciprocal effects on APP β- and α-cleavage in APP/BACE^(single_copy)cells as well, confirming that our editing strategy has reciprocaleffects on β/α cleavage (FIG. 8G). Further experiments using an APP-C99construct (wild-type and truncated construct mimicking theCRISPR-product, APP-659) precludes an effect of the sgRNA onAPP-γ-cleavage (FIGS. 9A-9C), indicating that our editing strategy isselectively affecting APP β-cleavage. Collectively, the available datastrongly suggest that our gene editing strategy targeting the APPC-terminus is favorably manipulating the amyloid pathway by attenuatingAPP β-cleavage, while reciprocally up-regulating protective α-cleavage.

Off-target analysis and effect of APP C-terminus editing on neuronalphysiology—Off-target effects of CRISPR/Cas9, due to unwanted editing ofDNA-stretches resembling the targeted region, are a concern. Towardsthis, we asked if our mouse and human APP-sgRNA were able to edit thetop five computationally predicted off-target sites (FIG. 10A; also seeTable 3). No editing was seen using T7 endonuclease assays (FIGS.10B-10E). Though APP null mice are viable, there is compensation by thetwo APP homologues APLP1 and 2 that undergo similar processing as APP(15,16). APLP1 and 2 were not amongst the top 50 predicted off-targetsites, as their corresponding sgRNA-target sites were substantiallydifferent from APP (see sequences in FIG. 10F). For further assurancethat our sgRNA was not editing the APP homologues, we performed specificoff-target TIDE (Tracking of Indels by DEcomposition) analyses (17) oncells carrying the sgRNA. As shown in FIG. 10G, TIDE analyses showed noediting of APLP 1/2 by the sgRNA.

APP has known physiologic roles in axon growth and signaling (18). Asnoted above, the N-terminus of APP—thought to play roles in axon growthand differentiation—is entirely preserved in our setting. The C-terminalAPP intracellular domain (AICD) has been implicated in genetranscription, though the effect appears to be both physiologic andpathologic (19,20). To examine potential deleterious effects of editingthe extreme C-terminus of APP, we turned to cultured hippocampal neuronswhere various parameters like neurite outgrowth and synapticstructure/function can be confidently evaluated. To study pre-synapsestructure and neuronal activity, we generated AAV9 viruses carrying themo-APP-sgRNA and Cas9, tagged with GFP and HA respectively (see vectordesign in FIG. 3A) that transduced almost all cultured neurons (FIG. 3Band data not shown). In blinded analyses, we found no significant effectof the mo-APP-sgRNA on neurite outgrowth, axon-length, synapticorganization, or neuronal activity (FIGS. 3C-3G). We reason that thelack of deleterious effects upon editing is likely because: 1) most ofthe APP molecule remains intact after editing; 2) the APP homologuesAPLP1/2—that undergo similar processing as APP, generate CTFs, and areknown to compensate for APP function—remain unedited; and 3)APP-cleavage is not entirely blocked by our approach.

Editing of APP C-terminus in vivo and mechanistic details of APP β/αmanipulation—Next we asked if the APP-sgRNA could edit endogenous APP inmouse brains. Injection of the AAV9s into mouse hippocampi (FIG. 4A) ledto efficient transduction of both sgRNA and Cas9 in dentate neurons(86.87±2.83% neurons carrying the sgRNA also had Cas9; seerepresentative images in FIG. 4B). Immunostaining of transduced neuronswith the APP Y188 antibody showed attenuated staining, suggestingediting of endogenous APP in vivo (FIGS. 4C and 4D). To achieve a morewidespread expression of the sgRNA and Cas9 in mouse brains—and alsoevaluate editing by biochemistry—we injected the viruses into theventricles of neonatal (P0) mice and examined the brain after 2-4 weeks(FIG. 4E). Previous studies have shown that when AAVs are injected intothe ventricles of neonatal mice, there is widespread delivery oftransgenes into the brain—also called somatic transgenesis (21,22).Indeed, APP Y188 immunostaining was attenuated in cortical regions (FIG.4F) and immunoblotting with the Y188 antibody also showed a decreasedsignal (FIG. 4G); indicating that the APP-sgRNA can edit APP in vivo.

To determine the mechanism by which the APP-sgRNA manipulates theamyloid pathway, we used a “CRISPR-mimic” truncated APP construct(APP-659) that is the major post-editing translational product in bothmouse and human cells (see FIG. 2H, FIG. 6E, and FIG. 7D). Using ourBifC assay (10), we first asked if the CRISPR-mimic APP-659 interactedwith BACE-1. APP-659/BACE-1 approximation was greatly attenuated incultured neurons (FIG. 5A), along with a decrease in β-CTF generation(FIG. 5B). Next we visualized axonal and dendritic transport of APP-WTand APP-659. Although there were minor changes (FIGS. 11A-11C and Table4), it seems unlikely that such small transport perturbations would leadto the dramatic attenuation of β-cleavage and Aβ-production seen in ourexperiments.

The CRISPR-edited segment of APP contains the residues T668 andY682-Y687 (YENPTY motif, see FIG. 5C; also present in APLP1/2), thathave been reported to play a role in A13 production (12, 23, 24).Specifically, APP phosphorylated at T668 has been reported to colocalizewith BACE-1 in endosomes (23), and the YENPTY motif is known to mediateAPP internalization from the plasma membrane (25). Examining the effectsof these residues in APP/BACE-1 BifC assays, we saw that the extent ofAPP/BACE-1 attenuation by the YENPTY mutation strongly resembled thedecrease in fluorescence complementation by the APP-659 construct (FIG.5D). A prediction from these experiments is that endocytosis of theCRISPR-mimic APP from the cell surface should be attenuated; and indeed,this was the case in internalization assays (FIG. 5E). Similar resultswere seen with an “APP-659-GG” construct that more closely resembles themost common post-editing translational product of our sgRNA (FIGS.12A-12C; also see post-editing products from human cells in FIG. 2H andFIG. 6E).

Collectively, the data suggest that our gene-editing approach does nothave a major effect on post-Golgi trafficking of APP, but attenuates itsendocytosis from cell surface, and consequently, its interaction withBACE-1 in endosomes—though we cannot exclude a direct effect of editingon APP/BACE-1 interaction. This is also consistent with previous studiesshowing that surface APP is internalized into endosomes, where it iscleaved by BACE-1 (26-29). Since most of the APP α-cleavage is thoughtto occur at the cell surface (30), this may also explain why thenon-amyloidogenic pathway is enhanced by our approach.

Using CRISPR/Cas9 technology, herein we provide conceptual proof for astrategy that selectively edits the C-terminus of APP and alters thebalance of APP-cleavage—attenuating β-cleavage and Aβ, whileupregulating neuroprotective α-cleavage. The N-terminus of APP—known toplay physiologic roles—is unaffected, along with the compensatory APPhomologues APLP1/2. No deleterious effects were seen in neurophysiologicparameters. Without wishing to be bound by any particular theory, ourstrategy likely works by editing the terminal YENPTY motif in APP thatis responsible for its internalization, subsequent APP/BACE-1association, and initiation of the amyloidogenic pathway; whileretention of APP at the plasma membrane may facilitate the upregulationof APP α-cleavage.

APP processing is regulated by α-, β-, and γ-secretases; and the variouscleavage products may play physiological functions that are not fullyunderstood (31,32). Previous studies suggest that in vivo deletion ofthe APP C-terminus blocks APP β-cleavage without obvious effects onneuroanatomy, behavior and neuronal activity in adult mice (13).Notably, the APP homologues APLP 1/2 also have YENPTY motifs(15,16)—that can presumably undergo endocytosis and protein-proteininteractions—and are expected to compensate for the loss of theC-terminus. The precise reasoning behind enhanced α-cleavage is unclear.We propose that retention of APP at the plasma membrane might beresponsible, but we cannot rule out other causes, including off-targeteffects, and further detailed studies may provide clarity.

Methods

Constructs, antibodies and reagents—For transient co-expression ofCRISPR/Cas9 components, APP sgRNA nucleotides were synthesized andcloned into pU6-(Bbs1)_CBh-Cas9-T2A-mCherry vector at Bbs1 site. Forviral transduction, a dual vector system was used to deliver CRISPR/Cas9components using AAV9 (33). For making the AAV9 vectors, the APP sgRNAwas cloned into pAAV9-U6sgRNA(SapI)_hSyn-GFP-KASH-bGH vector at Sap1site. The CRISPR/Cas9 stable cell lines were generated by lentivirusinfection as follows. The APP sgRNA was cloned into lentiCRISPR v2vector at Bbs1 site to produce lentivirus (34). For making APP deletionsand relevant constructs, the human APP659 truncation was PCR amplifiedand cloned at Hind3 and Sac2 sites of pVN to generate pAPP659:VN. TheBBS-APP659 was PCR amplified and cloned into pBBS-APP:GFP at Hind3 andSac2, replacing BBS-APP, to generate pBBS-APP659:GFP. ThepBBS-APP^(YENFTY):GFP was generated by site directed mutagenesis frompBBS-APP:GFP. The pAPP^(T668A):VN and pAPP^(T668A+YENPTY):VN weregenerated by site directed mutagenesis from pAPP:VN andpAPP^(YENPTY):VN. Antibodies used were as follows: APP Y188 (ab32136;Abcam), APP 22C11 (MAB348; Millipore), APP 6E10 (803001; BioLegend), APPM3.2 (805701; BioLegend), APP 2E9 (MABN2295; Millipore), APP CT20(171610; Millipore), sAPP0 (18957; IBL) BACE-1 (MAB931; R&D), GAPDH(MA5-15738, ThermoFisher), GFP (ab290, Abeam), GFP (A10262, Invitrogen),HA (901513, BioLegend), VAMP2 (104211, Synaptic Systems). Reagents wereas follows: γ-secretase inhibitor BMS-299897 (Sigma), and Rho Kinase(ROCK)-inhibitor H-1152P (Calbiochem).

Cell cultures, transfections, viral production/infections, andbiochemistry—HEK293 and neuro2a cells (ATCC) were maintained in DMEMwith 10% FBS. Cells were transfected with Lipofectamine™ 2000 andcollected 5 days after transfection for biochemical and immunostaininganalysis. All the studies involving primary neuron culture wereperformed in accordance with University of Wisconsin guidelines. Primaryhippocampal neurons were obtained from postnatal (P0-P1) CD1 mice(either sex), and transiently transfected using Lipofectamine™ 2000 orAmaxa™ 4D system (Lonza). Dissociated neurons were plated at a densityof 30,000 cells/cm² on poly-D-lysine-coated glass-bottom culture dishes(Mattek) and maintained in Neurobasal™/B27 medium with 5% CO₂. ForAPP/BACE-1 interaction and APP transport studies, DIV 7 neurons werecultured for ˜18-20 h after transfection. For spine density analysis,DIV7 neurons were transfected with Cas9, sgRNA and soluble marker, andcultured for 7 d before imaging. For testing the effect of CRISPR/Cas9on neuronal development, neurons were electroporated with the respectiveconstructs before plating using an Amaxa™ 4D-Nucleofector™ system withthe P3 Primary Cell 4D-Nucleofector™ X kit S and program CL-133.

For western blotting, pre-synapse analyses and electrophysiology, DIV7cultured neurons were infected with either AAV9-APP sgRNA-GFP (2.24×10¹³Vg/ml) and AAV9-Cas9 (2.4×10″ Vg/ml), or AAV9-GFP (2.58×10¹³ Vg/ml) andAAV9-Cas9 at a multiplicity of infection (MOI) of 1.5×10⁵. Neurons wereanalyzed 7 days post-infection. Lentivirus was produced from HEK293FTcells as described (35). Briefly, HEK293FT cells (Life Technologies)were maintained in DMEM with 10% FBS, 0.1 mM NEAA, 1 mM sodium pyruvateand 2 mM Glutamine. Cells were transfected with lentiviral-target andhelper plasmids at 80-90% confluency. 2 days after transfection, thesupernatant was collected and filtered with 0.45 μm filter. Forexperiments with hESCs, cells were cultured on a Matrigel® substrate (BDBiosciences) and fed daily with TeSR-E8 culture media (StemCellTechnologies). When the cells were around 60-70% confluent, they wereinfected with a 50/50 mixture of TeSR-E8 (with 1.0 μM H-1152P) andlentivirus supernatant. After 24 h, the virus was removed, and the cellswere fed for 2 days (to recover). After 3 days, cells were treated with0.33 μg/mL of puromycin for 72 h to select for virally-integrated hESCs.For HEK and neuro2a cell lines, cells were infected with the lentiviruscarrying APP-sgRNA and Cas9 for 24 h. And then cells were fed for 1 dayto recover. After 2 days, cells were treated with 1 μg/mL of puromycinfor 72 h to select for virally-integrated cells.

Human NPCs were generated as has been described previously (36), usingmanual rosette selection and Matrigel® (Corning) to maintain them.Concentrated lentiviruses express control-sgRNA or APP-sgRNA were madeas described previously (37), using Lenti-X™ concentrator (Clontech).The NPCs were transduced with either control-sgRNA or APP-sgRNA afterAccutase® splitting and were submitted to puromycin selection thesubsequent day. Polyclonal lines were expanded and treated withpuromycin for 5 more days before banking. Neuronal differentiations werecarried out by plating 165,000 cells/12 well-well in N2/B27 media(DMEM/F12 base) supplemented with BDNF (20 ng/mL; R&D) and laminin (1ug/mL; Trevigen).

For biochemistry, cell lysates were prepared in PBS+0.15% Triton™ X-100or RIPA supplemented with protease inhibitor cocktail, pH 7.4. Aftercentrifuging at 12,000 g for 15 min at 4° C., supernatants werequantified and resolved by SDS-PAGE for western blot analysis. For sAPPαand sAPPβ detection, cell culture medium was collected and centrifugedat 2,000 g for 15 min at RT. The supernatants were resolved by SDS-PAGEfor western blot analysis; band intensities were measured by ImageJ.Human Aβ40 and Aβ42 were detected using kits, according to themanufacturer's instructions (Thermo KHB3481 and KHB3544). Briefly,supernatants from H4^(single copy) cells or human iPSC derived neuronswere collected and diluted (×5 for H4 and ×2 for iPSC-neuron). Thediluted supernatants and the human A1340/42 detection antibodies werethen added into well and incubated for 3 h at RT with shaking. Afterwashing (×4), the anti-Rabbit IgG HRP solution was added and incubatedfor 30 min at RT. The stabilized Chromogen was added after washing (×4)and incubated for another 30 min at RT in the dark. After addition ofstop solution, absorbance at 450 nm was read using a luminescencemicroplate reader.

Developing a single-copy, stable APP/BACE-1 cell line—H4 tetOff FlpInempty clone was maintained in OptiMEM® with 10% FBS, 200 μg/mL G418 and300 μg/mL Zeocin. To generate an APP:VN/BACE-1:VC stable cell linecarrying single copies of APP and BACE-1, the expressing plasmid andpOG44 plasmids were transfected with Lipofectamine™ 2000. 2 days aftertransfection, cells were selected with 200 μg/mL hygromycin B and 200μg/mL G418 for 1 week. A monoclonal cell line with stable expression wasselected. H4 stable cell lines were then infected with the lentiviruscarrying APP-sgRNA and Cas9, as described above. After 24 h, the viruswas removed, and cells were fed for 1 day to recover. After 2 days,cells were treated with 0.7 μg/mL of puromycin for 72 h to select forvirally-integrated cells.

Generation of the APPLondon (V717I) knock-in iPSC line—CRIPSR/Cas9 wasused to knock in the APP V717I mutation (APPLon) into a commerciallyavailable control human iPSC line IMR90 (clone 4, WiCell). sgRNAstargeting Exon17 of APP were designed using the CRISPR design toolcreated by Feng Zhang's lab and subcloned into the MLM3636 vector(AddGene). Efficacy of multiple sgRNAs was first assessed in HEK293cells (Geneart™ Genomic Cleavage Detection Kit, Life Technologies). ThessDNA HDR template was designed to include a silent CRISPR blockingmutation at the PAM site of most efficacious sgRNA in addition to theAPPLon mutation. sgRNA, Cas9-2A-mCherry (generously provided by HynekWicterle), and ssDNA HDR template were electroporated (LonzaNucleofector™) into feeder-free IMR90 iPSCs, followed by cell sorting onmCherry signal and plating at low density on MEFs (MTI-GlobalStem).Individual clones were manually picked into a 96 well format,subsequently split into duplicate plates, one of which were used togenerate gDNA as had been done previously³⁸. For each clone, exon 17 ofAPP was amplified and initially screened by restriction digest for thepresence of a de novo BclI site introduce by the APPLon mutation. Sangersequencing was used to confirm the mutation, and successful knockinclones were expanded and banked. Potential off-target effects ofCRISPR/Cas9 cleavage were analyzed by Sanger sequencing of the top 5predicted off-target genomic locations, which demonstrated a lack ofindels for multiple clones. Clone 88 was picked for future studies.

Immunofluorescence, microscopy/image analysis, APP trafficking andendocytosis assays—For immunostaining of endogenous APP or VAMP2, cellswere fixed in 4% PFA/sucrose solution in PBS for 10 min at roomtemperature (RT), extracted in PBS containing 0.2% Triton™ X-100 for 10min at RT, blocked for 2 h at RT in 1% bovine serum albumin and 5% FBS,and then incubated with rabbit anti-APP (1:200) or mouse anti-VAMP2(1:1000) diluted in blocking buffer for 2 h at RT. After removal ofprimary antibody, cells were blocked for 30 min at RT, incubated withgoat anti-rabbit (Alexa Fluor 488) or goat anti-mouse (Alexa Fluor® 594)secondary antibody at 1:1000 dilution for 1 h at RT and then mounted forimaging. z-stack images (0.339 μm z-step) were acquired using aninverted epifluorescence microscope (Eclipse Ti-E) equipped with CFI SFluor VC 40× NA 1.30 (Nikon). An electron-multiplying charge-coupleddevice camera (QuantEM: 512SC; Photometrics) and LED illuminator(SPECTRA X; Lumencor) were used for all image acquisition. The systemwas controlled by Elements software (NIS Elements Advanced Research).z-stacks were subjected to a maximum intensity projection. For APP Y188staining, the average intensity of single cell body (neuro2A, HEK293 andneurons) or the whole colony (hESCs) was quantified. All the images wereanalyzed in Metamorph® and ImageJ.

Spine density experiments were done as described previously (39).Briefly, DIV 7 neurons were transfected with desired constructs for 7days, and secondary dendrites were selected for imaging. z-stack imageswere captured using a 100× objective (0.2 μm z-step) and subjected to amaximum intensity projection for analysis. For the APP/BACE-1complementation assay, DIV 7 neurons were transfected with desiredconstructs for ˜15-18 h and fixed. z-stack images were captured using a40× objective (0.339 μm z-step) and subjected to a maximum intensityprojection. The average intensity within cell bodies was quantified.

For trafficking studies in axons and dendrites, imaging parameters wereset at 1 frame/s and total 200 frames. Kymographs were generated inMetaMorph®, and segmental tracks were traced on the kymographs using aline tool. The resultant velocity (distance/time) and run length datawere obtained for each track, frequencies of particle movements werecalculated by dividing the number of individual particles moving in agiven direction by the total number of analyzed particles in thekymograph, and numbers of particles per minute were calculated bydividing the number of particles moving in a given direction by thetotal imaging time.

APP endocytosis assay was done as described previously (40). Cellsexpressing APP-GFP, APP659-GFP, untagged APP or untagged APP-659-GG werestarved with serum-free medium for 30 min and incubated with anti-APP(22C11) in complete medium with 10 mM HEPES for 10 min. And then, cellswere fixed, permeablized and immunostained for 22C11. The mean intensityof 22C11 along plasma membrane was calculated by dividing the totalintensity along plasma membrane (=intensity of whole cell−intensity ofcytoplasm) with area of plasma membrane (=area of whole cell−area ofcytoplasm). The ratio of mean intensities between plasma membrane andcytoplasm was quantified.

Stereotactic injection of AAV9s into the mouse brain and histology—Allthe animal procedures were performed in accordance with University ofWisconsin guidelines. In vivo injection and immunofluorescence stainingwas done as described previously (41). Briefly, 1.5 μl of 1:2 AAV9mixture of AAV9-APP sgRNA-GFP (or AAV9-GFP) and AAV9-Cas9 was injectedinto the dentate gyrus (−2.0, ±1.6, −1.9) of 8-week old male C57BL/6mice (either sex). 2-weeks after surgery, the mice were sacrificed bytrans-cardiac perfusion of saline, followed by 4% PFA. The brains weredissected, post-fixed with 4% PFA overnight, immersed in 30% sucroseuntil saturation, and sectioned at 40 μm. Sections were immunostainedwith the following antibodies: mouse anti-HA (1:1000, BioLegend, clone16B12), chicken anti-GFP (1:1000, Invitrogen, polyclonal) and rabbitanti-APP (1:200, Abcam, clone Y188). Images were acquired using ZeissLSM800 confocal microscope. Average intensities of APP staining in cellbodies was quantified using Metamorph®.

Intracerebroventricular injections and histology—All animal procedureswere approved by the Mayo Institutional Animal Care and Use Committeeand are in accordance with the NIH Guide for Care and Use of Laboratoryanimals. Free hand bilateral intracerebroventricular (ICV) injectionswere performed as previously described (42) in C57BL/6 mouse pups. Onpost-natal day 0, newborn pups were briefly cryoanesthetized on iceuntil no movement was observed. A 30-gauge needle attached to a 10 μlsyringe (Hamilton) was used to pierce the skull of the pups justposterior to bregma and 2 mm lateral to the midline. The needle was heldat a depth of approximately 2 millimeters, and 2 μl of a mixture of AAV9viruses (ratio 1:2 of AAV9-APP sgRNA-GFP or AAV9-GFP+ AAV9-Cas9) wereinjected into each cerebral ventricle. After 5 minutes of recovery on aheat pad, the pups were returned into their home cages. Mice weresacrificed 15 days after viral injection. Animals were deeplyanesthetized with sodium pentobarbital prior to transcardial perfusionwith phosphate buffered saline (PBS), and the brain was removed andbisected along the midline. The left hemisphere was drop-fixed in 10%neutral buffered formalin (Fisher Scientific, Waltham, Mass.) overnightat 4° C. for histology, whereas the right hemisphere of each brain wassnap-frozen and homogenized for biochemical analysis. Formalin fixedbrains were embedded in paraffin wax, sectioned in a sagittal plane at5-micron thickness, and mounted on glass slides. Tissue sections werethen deparaffinized in xylene and rehydrated. Antigen retrieval wasperformed by steaming in distilled water for 30 min, followed bypermeabilization with 0.5% Triton™-X, and blocking with 5% goat serumfor 1 hour. Sagittal sections were then incubated with primary anti-GFPantibody (1:250, Ayes, chicken polyclonal) and anti-APP antibody (1:200,Abcam, clone Y188) overnight at 4° C. Sections were incubated with thesecondary antibodies Alexa Fluor® 488-goat anti-chicken and AlexaFluor®568-goat anti rabbit (1:500, Invitrogen) for 2h at roomtemperature. Sections were washed and briefly dipped into 0.3% SudanBlack in 70% ethanol prior to mounting.

Electrophysiology—A coverslip with cultured cells at a density of 60,000cells/cm² was placed in a continuously perfused bath, viewed underIR-DIC optics and whole-cell voltage clamp recordings were performed(−70 mV, room temp.). The extracellular solution consisted of (in mM):145 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES and 10 dextrose, adjustedto 7.3 pH with NaOH and 320 mOsm with sucrose. Whole-cell recordingswere made with pipette solutions consisting of (in mM) 140 KCl, 10 EGTA,10 HEPES, 2 Mg2ATP and 20 phosphocreatine, adjusted to pH 7.3 with KOHand 315 mOsm with sucrose. Excitatory synaptic events were isolated byadding 10 μM bicuculline to block GABA (subscript A) receptors.Miniature synaptic events were isolated by adding 100 nM tetrodotoxin toprevent action potentials. mEPSCs were detected using thetemplate-matching algorithm in Axograph X, with a template that had 0.5ms rise time and 5 ms decay. Statistics were computed using theStatistics Toolbox of Matlab.

T7 Endonuclease 1 Assay, Off-target, and ICE analyses—Genomic PCR wasperformed around each sgRNA target, and related off-target sites,following the manufacturer's instruction (using AccuPrime™ HiFi Taqusing 500 ng of genomic DNA). Products were then purified using Wizard®SV Gel and PCR Clear-Up System (Promega), and quantified using a Qubit®2.0 (Thermo Fischer). T7E1 assay was performed according tomanufacturer's instructions (New England Biolabs). Briefly, 200 ng ofgenomic PCR was combined with 24, of NEBuffer™ 2 (New England Biolabs)and diluted to 19 μL. Products were then hybridized by denaturing at 95°C. for 5 minutes then ramped down to 85° C. at −2° C./second. This wasfollowed by a second decrease to 25° C. at −0.1° C./second. Tohybridized product, 1 μL T7E1 (M0302, New England Biolabs) was added andmixed well followed by incubation at 37° C. for 15 minutes. Reaction wasstopped by adding 1.5 μL of 0.25M EDTA. Products were analyzed on a 3%agarose gel and quantified using a Gel Doc XR system (BioRad).Off-target sites were identified and scored using Benchling. The top 5off-target sites—chosen on the basis of raw score and irrespective ofbeing in a coding region—were identified and analyzed using T7E1 assayas previously described. For TIDE (43), PCR was performed on genomic DNAusing Accuprime™ Taq HiFi (Thermo Fischer) according to manufacturespecifications. Briefly, reactions were cycled at 2 min at 94° C.followed by 35 cycles of 98° C. for 30 seconds, 58° C. for 30 seconds,and 68° C. for 2 minutes 30 seconds and a final extension phase of 68°C. for 10 minutes. Products were then subjected to Sanger Sequencing andanalyzed using the TIDE platform. The primers used for TIDE analyses arelisted in Table 3. For analyses of indel after CRISPR editing withAPP670-sgRNA and APP676-sgRNA, the edited regions of genomic DNA werePCR amplified and subjected to Sanger Sequencing. The results wereanalyzed using the ICE platform.

Deep Sequencing Sample Preparation and data analysis—Genomic PCR wasperformed using AccuPrime™ HiFi Taq (Life Technologies) followingmanufacturer's instructions. About 200-500 ng of genomic DNA was usedfor each PCR reaction. Products were then purified using AMPure® XPmagnetic bead purification kit (Beckman Coulter) and quantified using aNanodrop2000. Individual samples were pooled and run on an Illumina®HiSeq2500 High Throughput at a run length of 2×125 bp. A custom pythonscript was developed to perform sequence analysis. For each sample,sequences with frequency of less than 100 reads were filtered from thedata. Sequences in which the reads matched with primer and reversecomplement subsequences classified as target sequences. These sequenceswere then aligned with corresponding wildtype sequence using globalpairwise sequence alignment. Sequences that were misaligned through gapsor insertions around the expected cut site were classified as NHEJevents. The frequency, length, and position of matches, insertions,deletions, and mismatches were all tracked in the resulting alignedsequences.

Statistical analysis—Statistical analysis was performed and plottedusing Prism software. Student's t-test (unpaired, two-tailed) was usedto compare two groups. One-way ANOVA test was used to compare multiplegroups, following with Tukey multiple comparison test of every pair. AP-value <0.05 was considered significant.

TABLE 3  PCR Primers used for on- and off- targetgenomic loci amplification SEQ SEQ Forward primer ID Reverse primer IDsequence (5′-3′) NO: sequence (5′-3′) NO: Mouse APP AGGAACGGAGTGACCT 21TTCCTCCATGGTAACC 22 (659) GTTTCC ACGCAT Human APP TGGGGAAGCCACATGT 23ATGTTTTGGTGGGCCA 24 (659) TGTACA TTTGGT Human APP AAATTATGGGTGTTCT 25ACTTGTGTTACAGCAC 26 (670; 676) GCAATCTTGG AGCTGTC Mouse OT1GCCCTCCAGAAGTATT 27 GTCAGGGCCTTGCTCT 28 GGCTT ACAAA Mouse OT2CGCAAAAACTGGCTGC 29 TGTAGGCGCACATGCA 30 GTAT GAAG Mouse OT3CAGGTAGAGCGTGGAA 31 TGTGCGCATTAGGACC 32 ACTCA AGAT Mouse OT4CACCTGACAATGCTGT 33 AGACAAGGTCTGTCTC 34 CCCA CTTGC Mouse OT5CCAACTCTTTGCTTAG 35 ATCGTCCCTGGTGCAT 36 GGGC TCTC Human OT1GGAAAACCAGGTAGAG 37 TCTCTGGCTCGAGGGT 38 GGGG ACAT Human OT2CTGCATGCCATGGGTA 39 CAGGCTGTTTCGGGTC 40 GGTA CTT Human OT3AGACTCTTCTCCGATT 41 TCCAGCACGATCTGGT 42 CCAGC AGGC Human OT4AGTGCTTTTCTTTGCC 43 TGCTCGGGAGGTGTTT 44 TTTGCT CTAC Human OT5AACAAGGCAGCTCCTC 45 GACGTCAGAATTGAGG 46 AACT GTGGA Mouse CCAGCGGGATGAACTG 47 CCCAGGTCACCTTAAG 48 APLP1 GTAAGA GAGCAA Mouse GAGAGAGTTGGAGGCC 49 AACCACAGTGACAAGT 50 APLP2 TTGAGG GGCTCT Human GTGAATGCGTCTGTTC 51 GCTGCTGGGACTATCT 52 APLP1 CAAGGG GGGAAT Human TTTTAGGGGCTCGACC 53 TGCACTAATTTCCCAG 54 APLP2 TTCCAG GGCTCA

TABLE 4 Transport parameters of WT and APP659 Anterograde RetrogradeAnterograde Retrograde Run- velocity (μm/sec), velocity (μm/sec), %Anterograde % Retrograde % Stationary Run-length (μm) length (μm) mean ±SEM mean ± SEM Kinetics in axons APP659 53.88 ± 4.57 37.13 ± 4.36  8.97± 1.51 8.08 ± 0.31  6.8 ± 0.26 1.66 ± 0.03 1.52 ± 0.03 APPWT 57.84 ±2.22 34.37 ± 4.46 10.42 ± 4.24 10.44 ± 0.42  6.35 ± 0.26 1.97 ± 0.031.52 ± 0.03 Kinetics in dendrites APP659 37.81 ± 6.45 20.55 ± 6.21 41.64± 8.37  7.0 ± 0.48 6.94 ± 0.81 0.76 ± 0.05 0.83 ± 0.12 APPWT 45.83 ±4.58 24.81 ± 2.97 29.35 ± 5.63 8.12 ± 0.46 7.98 ± 0.94 0.94 ± 0.03  0.9± 0.07 ~115 APP659:GFP and ~130 APP:GFP vesicles analyzed in dendrites;~310 APP659:GFP and ~325 APP:GFP vesicles in axons (from 10-12 neuronsfrom 2 separate cultures.)

TABLE 5  APP sgRNAs targeting sequences SEQ sgRNA targeting sequenceID NO: Human APP 659 ATCCATTCATCATGGTGTGG 1 Human APP 670TGGACAGGTGGCGCTCCTCT 2 Human APP 676 GTAGCCGTTCTGCTGCATCT 4Mouse APP 659 ATCCATCCATCATGGCGTGG 6 Mouse APP 670 TGGAGAGATGGCGCTCCTCT7 Mouse APP 676 ATATCCGTTCTGCTGCATCT 9

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We claim:
 1. A method of treating Alzheimer's disease (AD) caused byformation of amyloid plaques composed of amyloid beta (AB) peptides,wherein the method comprises the steps of a) obtaining a gene-editingconstruct specific for the amyloid precursor protein (APP), wherein thegene-editing construct facilitates truncation of the APP C-terminus whencombined with a Cas9 nuclease, and b) delivering the gene-editingconstruct and a construct encoding the Cas9 nuclease to a patient inneed of AD therapy, wherein the APP molecule is truncated and productionof AB peptides is decreased in the patient's brain, wherein thetruncation of the APP C-terminus occurs at an APP residue selected fromthe group consisting of 659, 670, 676, and 686 relative to SEQ ID NO: 12(human) or SEQ ID NO: 14 (mouse).
 2. The method of claim 1, wherein thegene-editing construct comprises a gRNA sequence selected from the groupconsisting of SEQ ID Nos: 1-10.
 3. The method of claim 1, wherein thegene-editing construct and the construct encoding the Cas9 nuclease aredelivered in a composition comprising an adeno-associated viral vectorand a nanocarrier delivery vehicle.
 4. The method of claim 3, whereinthe composition is delivered intravenously or intrathecally.
 5. A methodof reducing the formation of amyloid plaques in a patient's brain,wherein the plaques comprise amyloid beta (AB) peptides, the methodcomprises the steps of a) obtaining a gene-editing construct specificfor the amyloid precursor protein (APP), wherein the gene-editingconstruct facilitates truncation of the APP C-terminus when combinedwith a Cas9 nuclease, and b) delivering the gene-editing construct and aconstruct encoding the Cas9 nuclease to a patient in need of AD therapy,wherein the APP molecule is truncated and production of AB peptides isdecreased in the patient's brain, wherein the truncation of the APPC-terminus occurs at an APP residue from the group consisting of 659,670, 676, and 686 relative to SEQ ID NO: 12 (human) or SEQ ID NO: 14(mouse).
 6. The method of claim 5, wherein the gene-editing constructcomprises a gRNA sequence selected from the group consisting of SEQ IDNOs: 1-10.
 7. The method of claim 5, wherein the gene-editing constructand the construct encoding the Cas9 nuclease are delivered in acomposition comprising an adeno-associated viral vector and ananocarrier delivery vehicle.
 8. The method of claim 5, wherein thecomposition is delivered intravenously or intrathecally.
 9. The methodof claim 1, wherein the gene-editing construct specific for the amyloidprecursor protein (APP) comprises a sequence encoding a gRNA specific toamyloid precursor protein (APP), and wherein a Cas9 nuclease/gRNAribonucleoprotein directs cleavage of the APP gene.
 10. The method ofclaim 5, wherein the gene-editing construct specific for the amyloidprecursor protein (APP) comprises a sequence encoding a gRNA specific toamyloid precursor protein (APP), and wherein a Cas9 nuclease/gRNAribonucleoprotein directs cleavage of the APP gene.